Our HIV vaccine strategy uses an initial immunization with a replicating adenovirus (Ad) vector carrying an HIV/SIV gene(s) followed by a booster immunization with an HIV/SIV envelope protein. The vectored vaccine replicates in epithelial cells that line mucosal inductive sites, thus eliciting strong, persistent cellular immunity at mucosal effector sites as well as in the blood. We have compared mucosal immunization routes to evaluate the biodistribution of the replicating vector, its persistence in the host, and its ability to elicit both systemic and mucosal immune responses. Using the SIV rhesus macaque model, we found that immunization with replication-competent Ad-SIV recombinants, regardless of the immunization route (either sublingual, intranasal/intratracheal, intravaginal, or intrarectal) led to expression of inserted genes first in macrophages in lung and rectal tissue, and subsequently in myeloid dendritic cells of the lung, with persistent expression in rectal tissue up to 25 weeks post-immunization. This targeting of macrophages and professional antigen presenting cells and the persistent expression provide potent immunogenicity, both systemically and mucosally. In line with the similar biodistribution, comparable SIV-specific immunity was elicited by all mucosal immunization routes. We have also shown in a study evaluating vaccines based on Tat and Envelope, that the replicating Ad vector elicits a spectrum of cytokine/chemokine responses, which contribute to elicitation of the adaptive immune responses. These are unaffected by the inserted genes. Additionally, because non-neutralizing antibody responses have been shown to be contributors to protective efficacy, we have undertaken studies of effector cells which mediate some of these responses. Recently we reported a study using SIV-infected rhesus macaques, and showed upon Gag stimulation of macaque PBMCs, NK cell responses were induced in SIV-controlling, but not non-controlling animals. These NK cell responses were dependent on antigen-specific IL-2 production by CD4+ central memory T cells. The NK cell activation was blocked by anti-IL-2 neutralizing antibody and by CD4+ T cell depletion which abrogated the Gag-specific responses. Among tissue-resident cells, splenic and circulatory NK cells displayed similar activation profiles, whereas liver and mucosal NK cells displayed a decreased activation profile, similar in SIV controlling and non-controlling macaques. Lack of T cell-dependent NK cell function was rescued in SIV non-controlling macaques through drug-mediated control of viremia. These results suggest that control of disease progression in SIV controlling macaques is associated with co-operation between antigen-specific CD4+ T cells and NK cell effector function and highlight the importance of such cell-to-cell cooperation in adaptive immunity.